Alex Christopher Engel

All Publications

• Cofactors Required for TLR7-and TLR9-Dependent Innate Immune Responses CELL HOST & MICROBE Chiang, C., Engel, A., Opaluch, A. M., Ramos, I., Maestre, A. M., Secundino, I., De Jesus, P. D., Nguyen, Q. T., Welch, G., Bonamy, G. M., Miraglia, L. J., Orth, A. P., Nizet, V., Fernandez-Sesma, A., Zhou, Y., Barton, G. M., Chanda, S. K. 2012; 11 (3): 306-318

  Abstract

  Pathogens commonly utilize endocytic pathways to gain cellular access. The endosomal pattern recognition receptors TLR7 and TLR9 detect pathogen-encoded nucleic acids to initiate MyD88-dependent proinflammatory responses to microbial infection. Using genome-wide RNAi screening and integrative systems-based analysis, we identify 190 cofactors required for TLR7- and TLR9-directed signaling responses. A set of cofactors were crossprofiled for their activities downstream of several immunoreceptors and then functionally mapped based on the known architecture of NF-κB signaling pathways. Protein complexes and pathways involved in ubiquitin-protein ligase activities, sphingolipid metabolism, chromatin modifications, and ancient stress responses were found to modulate innate recognition of endosomal nucleic acids. Additionally, hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) was characterized as necessary for ubiquitin-dependent TLR9 targeting to the endolysosome. Proteins and pathways identified here should prove useful in delineating strategies to manipulate innate responses for treatment of autoimmune disorders and microbial infection.

  View details for DOI 10.1016/j.chom.2012.02.002

  View details for Web of Science ID 000302050700011

  View details for PubMedID 22423970

  View details for PubMedCentralID PMC3310399

• Nucleic acid recognition by Toll-like receptors is coupled to stepwise processing by cathepsins and asparagine endopeptidase JOURNAL OF EXPERIMENTAL MEDICINE Ewald, S. E., Engel, A., Lee, J., Wang, M., Bogyo, M., Barton, G. M. 2011; 208 (4): 643-651

  Abstract

  Toll-like receptor (TLR) 9 requires proteolytic processing in the endolysosome to initiate signaling in response to DNA. However, recent studies conflict as to which proteases are required for receptor cleavage. We show that TLR9 proteolysis is a multistep process. The first step removes the majority of the ectodomain and can be performed by asparagine endopeptidase (AEP) or cathepsin family members. This initial cleavage event is followed by a trimming event that is solely cathepsin mediated and required for optimal receptor signaling. This dual requirement for AEP and cathepsins is observed in all cell types that we have analyzed, including mouse macrophages and dendritic cells. In addition, we show that TLR7 and TLR3 are processed in an analogous manner. These results define the core proteolytic steps required for TLR9 function and suggest that receptor proteolysis may represent a general regulatory strategy for all TLRs involved in nucleic acid recognition.

  View details for DOI 10.1084/jem.20100682

  View details for Web of Science ID 000289404800002

  View details for PubMedID 21402738

  View details for PubMedCentralID PMC3135342

• The yeast cell fusion protein Prm1p requires covalent dimerization to promote membrane fusion. PloS one Engel, A., Aguilar, P. S., Walter, P. 2010; 5 (5): e10593

  Abstract

  Prm1p is a multipass membrane protein that promotes plasma membrane fusion during yeast mating. The mechanism by which Prm1p and other putative regulators of developmentally controlled cell-cell fusion events facilitate membrane fusion has remained largely elusive. Here, we report that Prm1p forms covalently linked homodimers. Covalent Prm1p dimer formation occurs via intermolecular disulfide bonds of two cysteines, Cys-120 and Cys-545. PRM1 mutants in which these cysteines have been substituted are fusion defective. These PRM1 mutants are normally expressed, retain homotypic interaction and can traffic to the fusion zone. Because prm1-C120S and prm1-C545S mutants can form covalent dimers when coexpressed with wild-type PRM1, an intermolecular C120-C545 disulfide linkage is inferred. Cys-120 is adjacent to a highly conserved hydrophobic domain. Mutation of a charged residue within this hydrophobic domain abrogates formation of covalent dimers, trafficking to the fusion zone, and fusion-promoting activity. The importance of intermolecular disulfide bonding informs models regarding the mechanism of Prm1-mediated cell-cell fusion.

  View details for DOI 10.1371/journal.pone.0010593

  View details for PubMedID 20485669

  View details for PubMedCentralID PMC2868043

• Structure of sterol aliphatic chains affects yeast cell shape and cell fusion during mating. Proceedings of the National Academy of Sciences of the United States of America Aguilar, P. S., Heiman, M. G., Walther, T. C., Engel, A., Schwudke, D., Gushwa, N., Kurzchalia, T., Walter, P. 2010; 107 (9): 4170-5

  Abstract

  Under mating conditions, yeast cells adopt a characteristic pear-shaped morphology, called a "shmoo," as they project a cell extension toward their mating partners. Mating partners make contact at their shmoo tips, dissolve the intervening cell wall, and fuse their plasma membranes. We identified mutations in ERG4, encoding the enzyme that catalyzes the last step of ergosterol biosynthesis, that impair both shmoo formation and cell fusion. Upon pheromone treatment, erg4Delta mutants polarized growth, lipids, and proteins involved in mating but did not form properly shaped shmoos and fused with low efficiency. Supplementation with ergosterol partially suppressed the shmooing defect but not the cell fusion defect. By contrast, removal of the Erg4 substrate ergosta-5,7,22,24(28)-tetraenol, which accumulates in erg4Delta mutant cells and contains an extra double bond in the aliphatic chain of the sterol, restored both shmooing and cell fusion to wild-type levels. Thus, a two-atom change in the aliphatic moiety of ergosterol is sufficient to obstruct cell shape remodeling and cell fusion.

  View details for DOI 10.1073/pnas.0914094107

  View details for PubMedID 20150508

  View details for PubMedCentralID PMC2840153

• Membrane lysis during biological membrane fusion: collateral damage by misregulated fusion machines. The Journal of cell biology Engel, A., Walter, P. 2008; 183 (2): 181-6

  Abstract

  In the canonical model of membrane fusion, the integrity of the fusing membranes is never compromised, preserving the identity of fusing compartments. However, recent molecular simulations provided evidence for a pathway to fusion in which holes in the membrane evolve into a fusion pore. Additionally, two biological membrane fusion models-yeast cell mating and in vitro vacuole fusion-have shown that modifying the composition or altering the relative expression levels of membrane fusion complexes can result in membrane lysis. The convergence of these findings showing membrane integrity loss during biological membrane fusion suggests new mechanistic models for membrane fusion and the role of membrane fusion complexes.

  View details for DOI 10.1083/jcb.200805182

  View details for PubMedID 18852300

  View details for PubMedCentralID PMC2568015

• The plasma membrane proteins Prm1 and Fig1 ascertain fidelity of membrane fusion during yeast mating. Molecular biology of the cell Aguilar, P. S., Engel, A., Walter, P. 2007; 18 (2): 547-56

  Abstract

  As for most cell-cell fusion events, the molecular details of membrane fusion during yeast mating are poorly understood. The multipass membrane protein Prm1 is the only known component that acts at the step of bilayer fusion. In its absence, mutant mating pairs lyse or arrest in the mating reaction with tightly apposed plasma membranes. We show that deletion of FIG 1, which controls pheromone-induced Ca(2+) influx, yields similar cell fusion defects. Although extracellular Ca(2+) is not required for efficient cell fusion of wild-type cells, cell fusion in prm1 mutant mating pairs is dramatically reduced when Ca(2+) is removed. This enhanced fusion defect is due to lysis. Time-lapse microscopy reveals that fusion and lysis events initiate with identical kinetics, suggesting that both outcomes result from engagement of the fusion machinery. The yeast synaptotagmin orthologue and Ca(2+) binding protein Tcb3 has a role in reducing lysis of prm1 mutants, which opens the possibility that the observed role of Ca(2+) is to engage a wound repair mechanism. Thus, our results suggest that Prm1 and Fig1 have a role in enhancing membrane fusion and maintaining its fidelity. Their absence results in frequent mating pair lysis, which is counteracted by Ca(2+)-dependent membrane repair.

  View details for DOI 10.1091/mbc.e06-09-0776

  View details for PubMedID 17151357

  View details for PubMedCentralID PMC1783792

• The Golgi-resident protease Kex2 acts in conjunction with Prm1 to facilitate cell fusion during yeast mating. The Journal of cell biology Heiman, M. G., Engel, A., Walter, P. 2007; 176 (2): 209-22

  Abstract

  The molecular machines that mediate cell fusion are unknown. Previously, we identified a multispanning transmembrane protein, Prm1 (pheromone-regulated membrane protein 1), that acts during yeast mating (Heiman, M.G., and P. Walter. 2000. J. Cell Biol. 151:719-730). Without Prm1, a substantial fraction of mating pairs arrest with their plasma membranes tightly apposed yet unfused. In this study, we show that lack of the Golgi-resident protease Kex2 strongly enhances the cell fusion defect of Prm1-deficient mating pairs and causes a mild fusion defect in otherwise wild-type mating pairs. Lack of the Kex1 protease but not the Ste13 protease results in similar defects. Deltakex2 and Deltakex1 fusion defects were suppressed by osmotic support, a trait shared with mutants defective in cell wall remodeling. In contrast, other cell wall mutants do not enhance the Deltaprm1 fusion defect. Electron microscopy of Deltakex2-derived mating pairs revealed novel extracellular blebs at presumptive sites of fusion. Kex2 and Kex1 may promote cell fusion by proteolytically processing substrates that act in parallel to Prm1 as an alternative fusion machine, as cell wall components, or both.

  View details for DOI 10.1083/jcb.200609182

  View details for PubMedID 17210951

  View details for PubMedCentralID PMC2063940